CN113005086A - Application of epothilone D and Apol8 in regulation and control of neural stem cell directed neuron differentiation - Google Patents
Application of epothilone D and Apol8 in regulation and control of neural stem cell directed neuron differentiation Download PDFInfo
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Abstract
The invention relates to the technical field of neurology, and in particular relates to application of epothilone D and Apol8 in regulation and control of neural stem cell directed neuron differentiation. The invention discovers that epothilone D can promote the directional neuron differentiation of neural stem cells and inhibit the differentiation of the neural stem cells to astrocytes. Transplanting the epothilone D treated neural stem cells in the spinal cord injury area can promote the directional differentiation of the transplanted neural stem cells into neurons and participate in the formation of functional structures relay. Epothilone D regulates neural stem cell committed neuronal differentiation by up-regulating the Apol8 gene. The Apol8 gene can regulate the directional neuron differentiation of the neural stem cells. The discovery of new functions of Epothilone D and Apol8 in regulating and controlling the directional neuron differentiation of spinal cord neural stem cells provides a new target point for the treatment of spinal cord injury repair.
Description
Technical Field
The invention relates to the technical field of neurology, and in particular relates to application of epothilone D and Apol8 in regulation and control of neural stem cell directed neuron differentiation.
Background
Neural stem cells (neural stem cells) exist in the nervous system and have the potential to differentiate into neurons, astrocytes and oligodendrocytes. Neurons play an important role in functional reconstruction and nerve regeneration in central nervous system diseases. The differentiation of the neural stem cells to the neurons is regulated and controlled by various factors, and the enhancement of the directional regulation and control of the neural stem cells to the neurons has important significance for repairing central nerve injuries.
Spinal Cord Injury (SCI) repair is a significant challenge facing the field of regenerative medicine. The inhibitory microenvironment formed at the injury site following spinal cord injury hinders nerve regeneration, and therefore, promotion of axonal regeneration and neuronal differentiation is critical for spinal cord injury repair. Early studies showed that microtubule stabilizing agents have the effect of promoting axonal extension and are used in the treatment of spinal cord injury repair. Recent research shows that epothilone D (epothilone D) with microtubule stabilizing effect has simple structure, better water solubility and lower cytotoxicity, is easy to pass through blood brain barrier and not easy to diffuse, and can promote axon sprouting, inhibit fibrosis scar formation and improve motor function in the research of spinal cord injury. However, its regulatory role in spinal Neural Stem Cell (NSCs) differentiation is unclear.
Disclosure of Invention
The invention aims to provide application of epothilone D in regulation and control of differentiation of neural stem cells and directional neurons, and also provides application of Apol8 in regulation and control of differentiation of neural stem cells and directional neurons.
In the process of research on the directional differentiation of spinal cord neural stem cells, the invention discovers that epothilone D can promote the directional neuron differentiation of the neural stem cells in-vitro differentiation culture; moreover, after the spinal cord neural stem cells treated by the epothilone D are transplanted to a spinal cord injury area, the capacity of directionally differentiating the neurons is obviously enhanced, the spinal cord neural stem cells can participate in the formation of a functional structure relay, and meanwhile, the epothilone D can also inhibit the spinal cord neural stem cells from differentiating to astrocytes. Through further RNA-seq studies, epothilone D treatment was found to up-regulate the expression of Apol8 gene. Further, functional verification experiments on the Apol8 gene show that the overexpression of the Apol8 gene can promote the directional differentiation of spinal cord neural stem cells to neurons, and the inhibition of the expression of the Apol8 gene can inhibit the directional differentiation of the spinal cord neural stem cells to neurons.
Based on the above findings, the present invention specifically provides the following technical solutions:
the invention provides application of epothilone D in regulation and control of neural stem cell directed neuron differentiation.
Preferably, the neural stem cell is a spinal cord neural stem cell.
Specifically, the method can regulate the differentiation of the neural stem cell committed neurons to promote the differentiation of the spinal neural stem cell committed neurons.
The above-described regulation may be in vitro culture or in vivo regulation.
The invention provides an application of epothilone D in inhibition of differentiation from spinal neural stem cells to astrocytes.
The invention also provides application of the epothilone D in promoting differentiation of neural stem cells in spinal cord injury areas into directional neurons or formation of functional structures.
Preferably, the neural stem cells are transplanted spinal neural stem cells.
Specifically, the above-mentioned applications may be: treating spinal cord neural stem cells by using epothilone D in vitro, and transplanting the treated spinal cord neural stem cells to a spinal cord injury area.
The invention provides application of epothilone D in preparation of a product for promoting differentiation of neural stem cells in spinal cord injury areas into directional neurons or formation of functional structures.
In particular, the product may, in use, be: treating spinal cord neural stem cells by using epothilone D in vitro, and transplanting the treated spinal cord neural stem cells to a spinal cord injury area.
The treatment of the spinal cord neural stem cells by using the epothilone D can be specifically realized by adding the epothilone D with the final concentration of 0.5-5 nM into an in-vitro culture medium of the spinal cord stem cells.
The invention also provides application of epothilone D in regulation and control of Apol8 gene expression.
The invention also provides application of epothilone D in regulation and control of neural stem cell directional neuron differentiation through up-regulation of Apol8 gene.
The invention also provides application of epothilone D in regulation and control of neural stem cell directional neuron differentiation in spinal cord injury regions through up-regulation of Apol8 gene.
Preferably, the neural stem cells are transplanted spinal neural stem cells.
According to the invention, after treating a spinal cord injury mouse by using epothilone D, RNA-seq analysis of spinal cord tissues discovers a new function of the Apol8 gene, and the Apol8 gene can regulate and control directional neuron differentiation of neural stem cells in vitro and in vivo, and promote repair of spinal cord injury and recovery of motor functions.
The invention provides an application of Apol8 protein, a coding gene thereof or a biological material containing the coding gene thereof in regulating and controlling the directional neuron differentiation of neural stem cells.
Preferably, the neural stem cell is a spinal cord neural stem cell.
The neural stem cell directed neuron differentiation regulation is in vitro regulation or in vivo regulation.
The invention provides an application of Apol8 protein, a coding gene thereof or a biological material containing the coding gene thereof in preparing a product for promoting spinal cord injury repair.
The invention provides an application of Apol8 protein, a coding gene thereof or a biological material containing the coding gene thereof in preparing a product for promoting the formation of a synaptic structure in a spinal cord injury region.
The invention provides application of Apol8 protein, a coding gene thereof or biological materials containing the coding gene thereof in preparing products for promoting recovery of motor functions after spinal cord injury.
The invention also provides an inhibitor of the Apol8 protein or the coding gene thereof or application of a biological material containing the inhibitor in inhibiting the directional neuron differentiation of spinal cord neural stem cells.
In the present invention, the biological material is a recombinant DNA, a vector or a host cell.
The recombinant DNA is an expression cassette, an encoding gene of Apol8 fusion protein and the like.
The vector is a plasmid vector, a virus vector and the like.
The host cell is a microbial cell or an animal cell.
In the invention, the inhibitor of Apol8 gene can inhibit the transcription or expression of the gene coding Apol8 protein, including but not limited to interfering RNA of Apol8 gene and the like.
The invention also provides a method for regulating and controlling the directional neuron differentiation of the neural stem cells in vitro, which comprises the following steps: treating the neural stem cell by using epothilone D, or regulating the expression level of Apol8 gene in the neural stem cell.
Preferably, the neural stem cell is a spinal cord neural stem cell.
Preferably, the neural stem cell is promoted to be differentiated towards the neuron in vitro by treating the neural stem cell with epothilone D or by increasing the expression level of Apol8 gene in the neural stem cell.
Preferably, the epothilone D treatment is that the epothilone D with the concentration of 0.5-5 nM is used for treating the neural stem cells. The treatment is preferably to add 0.5-5 nM epothilone D to the neural stem cell in-vitro culture medium.
Epothilone D can be added to any in vitro differentiation medium suitable for neural stem cells, for example: DMEM/F12 medium with B27 added, and the like.
In the present invention, the neural stem cell may be derived from any mammal, including but not limited to mouse, rat, guinea pig, gerbil, rabbit, cat, dog, cow, horse, pig, sheep, and human, etc.
In the invention, the Apol8 protein is preferably mouse (Mus musculus) Apol8 protein, the sequence of the mouse Apol8 protein is shown as SEQ ID NO.1, and the nucleotide sequence of the Apol8 gene is shown as SEQ ID NO. 2.
The invention has the beneficial effects that: the invention discovers that epothilone D can promote the differentiation of neural stem cells into directional neurons, inhibit the differentiation of spinal neural stem cells into astrocytes, induce transplanted spinal neural stem cells into directional differentiation neurons in a spinal cord injury area and participate in the formation of functional structures relay. The invention further discovers that epothilone D regulates the directional neuronal differentiation of neural stem cells by up-regulating Apol8 gene. Functional verification experiments on the Apol8 gene show that the Apol8 gene can regulate the directional neuron differentiation of the neural stem cells, the overexpression of the Apol8 gene can promote the directional neuron differentiation of the spinal neural stem cells, and the inhibition of the expression of the Apol8 gene can inhibit the directional neuron differentiation of the spinal neural stem cells. The discovery of new functions of epothilone D and Apol8 in regulation of spinal cord neural stem cell directed neuron differentiation and the like provides a new target point for the treatment of spinal cord injury repair.
Drawings
Fig. 1 shows that epothilone D promotes neural stem cell directed neuronal differentiation and realy formation in example 1 of the present invention, wherein a is an immunofluorescence experiment that indicates that epothilone D promotes spinal neural stem cells to differentiate towards neurons and inhibits differentiation towards astrocytes; b and C are statistics chart of differentiation ratio of neurons and astrocytes in A; d and E are quantitative PCR detection for the influence of epothilone D on spinal cord neural stem cell differentiation in the spinal cord neural stem cell differentiation process; f and G are Western-blot to detect the influence of epothilone D on spinal cord neural stem cell differentiation in the spinal cord neural stem cell differentiation process; h is pseudorabies virus tracing experiment, which shows that spinal cord neural stem cells after directional domestication of epothilone D can be differentiated into mature neurons and participate in the formation of functional structure relay after transplantation; i and J are respectively the statistical results of the synaptic transmission ratio of the normal tissue and the damaged area in H, wherein Control in the figure represents a Control group, Epothilone D represents an Epothilone D treatment group, NSCs represents neural stem cell transplantation without Epothilone D treatment, and Epothilone D-NSCs represents neural stem cell transplantation treated by Epothilone D.
FIG. 2 shows that epothilone D promotes spinal neural stem cell-directed neuronal differentiation via Apol8 in example 2 of the present invention, wherein A is RNA-Seq results to find epothilone D regulated up-regulated expression genes; b is the effect of overexpression of Apol8 in spinal cord neural stem cells or interference of Apol8 by RNAi on spinal cord neural stem cell differentiation, where CD511B represents control spinal cord neural stem cells infected with lentiviral CD511B vector not carrying Apol8, CD511B-Apol8 represents spinal cord neural stem cells over-expressed by Apol8, and Apol8-RNAi represents spinal cord neural stem cells interfering with Apol 8; c is after interfering with Apol8 in spinal cord neural stem cells, further observe the influence of Epothilone D on its differentiation, in the figure, Control represents normal differentiation group, Epothilone D represents Epothilone D treatment group, Apol8-RNAi + Epothilone D represents Epothilone D treatment group after RNAi interferes with Apol 8.
FIG. 3 is the functional reconstruction after transplantation of neural stem cells over-expressed by Apol8 into mature neurons participating in spinal cord injury in example 2 of the present invention, wherein A is the differentiation into neurons in the injury region after Apol8-NSC transplantation; b is Apol8-NSC transplantation, which promotes the formation of synaptic structures, wherein the arrows indicate that synaptic structures are formed around the transplanted cells (GFP-labeled); c is a functional biological material LOCS loaded with Apol8-NSC, and can effectively improve the motor function of a mouse with spinal cord full-transection injury after transplantation; in the figure, Apol8-NSCs represents transplanted Apol8 gene overexpressed spinal cord neural stem cells, NSCs represents transplanted Apol8 gene unexpressed spinal cord neural stem cells, LOCS + NSCs represents functional biological material LOCS loaded Normal NSC transplantation, LOCS + Apol8-NSCs represents functional biological material LOCS loaded Apol8-NSC transplantation, Normal mouse represents Normal mouse, and SCI represents spinal cord full-transection injured mouse.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1 epothilone D promotes spinal neural stem cell directed neuronal differentiation
In this example, the function of epothilone D was analyzed by in vitro neural stem cell differentiation assay and in vivo transplantation assay, respectively, using the following specific methods:
1. in vitro neural stem cell differentiation experiment
(1) Isolating primary spinal cord neural stem cells of suckling mice with C57BL/6N within 12 hours of birth and performing spheroidicity proliferation culture in vitro (1 mL of non-essential amino acid, 1mL of sodium pyruvate, 1mL of Penicillin-streptomycin double antibody, 2mL of B27, 2mL of 30% glucose, 2 μ L of 1mg/mL of EGF, 2 μ L of 1mg/mL of bFGF, 7.32 μ L of heparin with the concentration of 0.05g/2mL is added in 100mL of DMEM/F12 culture medium);
(2) digesting the neural stem cell balls obtained in the step (1) into single cells, performing adherent culture by using an adherent culture medium overnight, replacing the culture medium the next day by using a differentiation culture medium (100mL of DMEM/F12 culture medium is added with 1mL of non-essential amino acid, 1mL of sodium pyruvate, 1mL of double antibody, 2mL of B27, 2mL of 30% glucose and 1mL of 3% glutamine), adding epothilone D (final concentration is 1nM) diluted by DMSO into the differentiation culture medium, performing differentiation culture for 6 days and 12 days, replacing the culture solution every other day, and collecting cells for Tuj-1 staining and MAP2 staining respectively.
Test groups: the medium was a differentiation medium supplemented with epothilone D to a final concentration of 1 nM.
Control group: the medium was a differentiation medium supplemented with an equal volume of DMSO.
The test group and the control group are changed every other day, and the epothilone D is added at present.
And (3) detecting the differentiation condition of the neural stem cells derived from the spinal cord of the mice under the action of the epothilone D by an immunofluorescence experiment. The results show that: under the differentiation culture condition of adding epothilone D, the differentiation proportion of neurons is remarkably improved, and the proportion of astrocytes is remarkably reduced (A-C in figure 1, and B and C are statistical results of A); the quantitative PCR detection of the gene expression conditions of neuron markers Tuj-1 and MAP2 shows that epothilone D can significantly improve the mRNA levels (D-E in figure 1) of Tuj-1 and MAP2, and indicates that epothilone D promotes spinal neural stem cells to differentiate towards neurons and inhibits the spinal neural stem cells from differentiating towards astrocytes; western-blot detection of levels of Tuj-1, MAP2 and GFAP proteins in spinal cord neural stem cell differentiation process shows that protein levels of Tuj-1 and MAP2 are remarkably improved, and GFAP protein levels are remarkably reduced (F-G in figure 1, G is a grey statistics result of F), which indicates that epothilone D can promote spinal cord neural stem cell directed neuron differentiation.
2. Transplantation of spinal neural stem cells treated with epothilone D
After the spinal cord neural stem cells are attached to the wall overnight, the proliferation culture medium is used for proliferation culture for 2 days, then retroviruses used in pseudorabies virus tracing experiments are used for marking the cells, epothilone D is added for in-vitro domestication culture treatment on the marked 3 rd day, then the cells are inoculated on LOCS biological materials for incubation overnight, and the cell transplant is transplanted to the full-transection injury part of the spinal cord the next day. Then, after 7 weeks after the spinal cord injury, pseudorabies virus, the second virus used in the pseudorabies virus tracing technology, is injected at the tail end of the spinal cord injury site, and finally, the materials are taken and stained after one week.
The pseudorabies virus tracing experiment is utilized to detect whether the transplanted neural stem cells are differentiated into mature neurons in the spinal cord injury region and participate in the formation of the functional structure relay, and the result shows that: after the spinal cord neural stem cells subjected to directional acclimation of epothilone D are transplanted, the spinal cord neural stem cells can be differentiated into mature neurons and participate in the formation of functional structures (H-J in figure 1).
In conclusion, in vitro differentiation experiments show that epothilone D can promote spinal neural stem cell directed neuron differentiation; through in vivo transplantation experiments of spinal cord neural stem cells, the spinal cord neural stem cells directionally domesticated by epothilone D can be differentiated into mature neurons in a spinal cord injury region and participate in the formation of functional structures relay after being transplanted.
Example 2 epothilone D regulates spinal neural stem cell committed neuronal differentiation by upregulating Apol8
A C57BL/6N mouse is subjected to 1mm total transverse spinal cord injury, then the C57BL/6N mouse is subjected to intraperitoneal injection administration once a day at a dose of 0.75 mg/kg of epothilone D for 5 days, spinal cord tissues of 2mm in total before and after an injury region are taken for RNA-seq, differential expression genes in the RNA-seq result are analyzed, and a total of 11 genes with significant difference in up-regulation expression in response to epothilone D regulation are found (A in figure 2), wherein the transcription level of an Apol8 gene is significantly improved, which indicates that epothilone D can up-regulate the expression of Apol 8.
In order to further analyze whether the Apol8 has the function of regulating and controlling the directional neuron differentiation of the spinal cord neural stem cells, Apol8 overexpression and RNAi interference on the spinal cord neural stem cells are respectively constructed, and the specific method comprises the following steps:
1. construction of Apol8 overexpressing spinal neural stem cells
The CDS region of mouse Apol8 (amino acid sequence is shown as SEQ ID NO.1, and gene sequence is shown as SEQ ID NO. 2) is constructed on a lentiviral vector CD511B, and then the lentivirus with Apol8 over-expression is obtained. And infecting adherent spinal cord neural stem cells of the mice by using lentiviruses to obtain Apol8 overexpression spinal cord neural stem cells.
2. RNAi interference Apol8 spinal cord neural stem cell construction
The mouse Apol8 gene sequence was submitted to a company designed siRNA sequence (SEQ ID NO. 3: CGTGCTTCAATATGTCTAA) and then transfected into adherent mouse spinal cord neural stem cells.
Spinal cord neural stem cells overexpressing Apol8 and interfered with RNAi were subjected to in vitro differentiation culture by the method of reference example 1, and normal spinal cord neural stem cells not overexpressing Apol8 and interfered with RNAi were used as a control. The proportion of neural stem cells differentiating to neurons is detected by adopting an immunofluorescence staining method, and the result shows that the neuron differentiation proportion can be remarkably improved by over-expressing Apol8 in spinal cord neural stem cells, the proportion of Tuj-1 positive cells is improved to 21.83 +/-2.68% from 7.44 +/-0.6% in a normal differentiation group, and the proportion is reduced to 4.40 +/-2.25% by interfering with Apol8 through RNAi. Meanwhile, the proportion of MAP2 mature neurons increased from 8.94 + -1.45% in the normal differentiation group to 23.72 + -3.16% in the Apol8 overexpression group, and decreased to 3.13 + -1.45% once Apol8 was interfered by RNAi (FIG. 2B), indicating that Apol8 can regulate spinal cord neural stem cell committed differentiation neurons.
Further, the RNAi interfering Apol8 spinal cord neural stem cells are treated by using epothilone D, and the experiment is divided into three groups, namely a normal differentiation group (not treated by epothilone D), an epothilone D treatment group (treated by epothilone D in normal spinal cord neural stem cells), and an epothilone D treatment group (treated by epothilone D in RNAi interfering Apol8 spinal cord neural stem cells) after RNAi interfering Apol 8.
Wherein, the treatment group of epothilone D after RNAi interference Apol8 is as follows: apol8-siRNA was transfected into adherent mouse spinal cord neural stem cells, and then differentiation medium containing 1nM epothilone D was replaced for differentiation culture.
The normal differentiation group is subjected to differentiation culture by using a differentiation medium without epothilone D.
The epothilone D treatment group was differential culture using a differentiation medium containing 1nM epothilone D.
The differentiation culture method was as in example 1.
The results of the above groups, which were examined for differentiation of neural stem cells by immunofluorescence staining, showed that, compared to the normal differentiation group, the epothilone D treated group had significantly increased the proportion of Tuj-1 and MAP2 positive cells and significantly decreased the proportion of GFAP astrocytes, whereas, once Apol8 in spinal cord neural stem cells was disturbed, epothilone D failed to increase the neuronal differentiation rate and did not decrease the proportion of astrocytes (fig. 2C), indicating that epothilone D promoted differentiation of spinal cord neural stem cells into neurons by Apol 8.
In conclusion, the overexpression experiment and RNAi experiment of the Apol8 gene prove that Epothilone D promotes the oriented neuron differentiation of spinal cord neural stem cells through Apol8, and the Apol8 gene has the function of regulating the oriented neuron differentiation of the spinal cord neural stem cells.
Further, the above-constructed Apol8 over-expressed neural stem cells (Apol8-NSC) were transplanted into the mouse spinal cord total transection injury region. The differentiation condition of transplanted neural stem cells in a spinal cord injury area and the improvement condition of mouse movement function recovery are detected by using a tissue immunofluorescence staining method and a BBB function scoring method, and the result shows that the differentiation ratio of transplanted normal spinal cord neural stem cells (spinal cord neural stem cells of which Apol8 gene is not overexpressed) to Tuj-1 positive cells in the spinal cord injury area is obviously lower than the differentiation ratio of transplanted Apol8-NSC (spinal cord neural stem cells of which Apol8 gene is overexpressed) to Tuj-1 positive cells, meanwhile, the transplanted normal NSCs cannot differentiate into mature neurons in the injury area, and Apol8-NSC can differentiate into mature neurons NeuN (A in figure 3) after transplantation, which shows that the transplanted Apol8-NSC can differentiate into more neurons in the injury area and part of the neurons can become mature neurons.
Immunofluorescent staining with neurofilaments and synaptic markers revealed some synaptic structures around the transplanted Apol8-NSC (fig. 3B), indicating that Apol8-NSC transplantation can promote the formation of synaptic structures. Meanwhile, a functional biological material LOCS (ordered collagen material) is used for carrying Apol8-NSC transplantation, and a BBB function scoring method is used for detecting the recovery condition of the motor function of the mouse, and the result shows that the Apol8-NSC transplantation group mouse has obvious motor function improvement (C in figure 3) compared with the normal NSC transplantation group mouse, and the Apol8-NSC transplantation can effectively improve the motor function of the mouse with the spinal cord total transection injury.
In conclusion, the neural stem cells over-expressed by the Apol8 transplanted in the spinal cord injury area can be differentiated into mature neurons and promote the formation of synaptic structures, thereby improving the recovery of motor functions of the mice with spinal cord full-transection injury.
In conclusion, the invention discovers for the first time that epothilone D can promote spinal neural stem cell directed neuron differentiation, and regulates the neuron differentiation process through Apol 8. The neural stem cells over-expressed by the Apol8 are more prone to be differentiated to mature neurons after spinal cord injury transplantation, so that the formation of a synaptic structure is promoted, and the motor function of a mouse is improved. The invention researches the function and action mechanism of epothilone D and Apol8 in regulating spinal cord neural stem cell differentiation, and provides a new target point for future treatment of spinal cord injury repair.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> institute of genetics and developmental biology of Chinese academy of sciences
Application of <120> epothilone D and Apol8 in regulation and control of neural stem cell directed neuron differentiation
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Claims (10)
1. Application of epothilone D in regulating and controlling directed neuron differentiation of neural stem cells.
2. The use according to claim 1, wherein said modulating differentiation of neural stem cell committed neurons promotes spinal neural stem cell committed neuron differentiation.
3. Use of epothilone D to inhibit differentiation of spinal neural stem cells into astrocytes.
4. The application of the epothilone D in promoting the differentiation of neural stem cells in the spinal cord injury area into directional neurons or the formation of a relay structure.
The use of Apol8 protein, its encoding gene or a biological material containing its encoding gene for regulating the differentiation of neural stem cells into targeted neurons;
preferably, the neural stem cell is a spinal cord neural stem cell.
Use of Apol8 protein, a gene encoding it or a biological material containing it for the preparation of a product for promoting the repair of spinal cord injuries.
Use of Apol8 protein, the gene encoding it or a biological material containing the gene encoding it for the preparation of a product intended to promote the formation of synaptic structures in areas of spinal cord injury.
Use of Apol8 protein, the gene encoding it or a biological material containing the gene encoding it for the preparation of a product intended to promote recovery of motor function following spinal cord injury.
Use of an inhibitor of Apol8 protein or its encoding gene or a biological material containing said inhibitor for inhibiting the directed neuronal differentiation of spinal neural stem cells.
10. A method for regulating and controlling the directional neuron differentiation of a neural stem cell in vitro, which is characterized in that the neural stem cell is treated by epothilone D, or the expression level of Apol8 gene in the neural stem cell is regulated and controlled.
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